FIGURE 8
FROM:
Construction of Escherichia coli K-12 in-frame, single-gene knockout mutants: the Keio collection
Tomoya Baba, Takeshi Ara, Miki Hasegawa, Yuki Takai, Yoshiko Okumura, Miki Baba, Kirill A Datsenko, Masaru Tomita, Barry L Wanner & Hirotada Mori
doi:10.1038/msb4100050
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PCR gene replacement strategy. (A) Gene targeting fragment encoding kanamycin resistance with short homology extensions (H1 and H2) is generated by PCR by using priming sites P1 and P2 (Step 1). Gene targeting fragment is introduced into E. coli K-12 BW25113 expressing the Red recombinase from pKD46 (Step 2). Kanamycin-resistant transformants are selected (Step 3). Transformants are verified by PCR (Step 4). (B) Elimination of the resistance cassette by use of the FLP recombinase plasmid pCP20 is expected to leave behind a 102-nt 'scar' encoding a 34-residue peptide (Step 1). The scar region is amplified and sequenced to be sure no mutations, especially 1-nt deletions (Datsenko and Wanner, 2000), were introduced (Step 2).
